This invention relates to coated substrates employing oriented layers of biological macromolecules (e.g., proteins) which have a preferential binding site that is capable of coupling with functional groups on the surface of a substrate. In particular, the present invention relates to substrates coated with mutant protein molecules having, at a select position in the amino acid sequencer either an amino acid or the side chain of the amino acid replaced to create the preferential binding site, which is in a select spatial relationship to a marker on the mutant protein. The mutant protein molecules are immobilized on a substrate in a desired pattern, which includes uniform immobilization over the entire surface of the substrate. Due to the spatial relationship between the marker and preferential binding site, the markers of the mutant protein molecules of the layers are in a select spatial relationship with both the substrate and the markers of the adjacent mutant protein molecules.
Surface-immobilized proteins have had a great impact in many fields of basic research, including many industrial and medical technologies. Most of the research in this area has been directed towards controlling the overall activity of hybrid biomaterials, for example, through reversibly affecting the surrounding matrix materials or by immobilizing related proteins in close proximity to provide multi-step enzyme processing.
Systems which utilize a layer of proteins coupled to inorganic carriers are well known for many purposes, including antigen or antibody purification, assaying and detecting biological reactions and sensor operations.
In such systems, the surface of the carrier is often treated to provide an intermediate coupling agent. For example, U.S. Pat. No. 3,652,761 to Weetall teaches utilizing silane coupling agents. U.S. Pat. No. 4,071,409 to Messing et al. teaches using polymeric isocyanates and discloses other coupling agents.
Such previous systems possessed limited usefulness. It was determined that a significant advance would be realized if the immobilization process itself specified assembly and regulated function, which allows selective control of molecular recognition events or molecular activity. Because many molecular recognition processes, such as protein-protein interactions, are controlled through specificity in complementary reactive surfaces, controlling the orientation of immobilized proteins can be a straightforward means of manipulating assembly and function. Similarly, molecular activity can also affected by orientation of the molecule on the substrate.
Proteins for use with this invention can have a marker such as a heme prosthetic group, an enzyme active site, a ligand or epitope binding site, a particular amino acid sequence or the like. The efficacy and usefulness of systems employing such proteins is greatly improved where the marker of the protein molecule is positioned in a particular, select relationship to the substrate surface and the markers of adjacent protein molecules.
Heretofore, random protein and marker positioning on a substrate surface could only be obtained because the vast majority of proteins have more than one reactive residue. These proteins are random positioning proteins. In other instances, some protein molecules have only one reactive residue on its surface and therefore will naturally be oriented relative to the substrate surface and adjacent proteins. However, the position of the one reactive residue limits the protein to be oriented in only one particular manner. Such a protein is not suitable if a different orientation is desired. Proteins that do not have a residue that is reactive with the coupling agent could not be previously utilized.
Random positioning of protein molecules on a substrate surface can be deleterious in a number of applications. For instance, in systems which utilize proteins that transfer electrons or electromagnetic radiation between adjacent proteins, random positioning of these protein molecules in the layer inhibits transfer between proteins and propagation through the layer as compared to systems where proteins are mutated so that the marker of the each immobilized protein molecule is in a select spatial relationship with both the substrate surface and the markers of adjacent protein molecules. Random positioning also can reduce the absorption of light by the layer of proteins as when each protein requires the light to be incident in a direction parallel to a light absorptive marker of the protein. Additionally, random positioning can adversely influence the binding and interaction characteristics of immobilized protein.
Industry has needs which can be satisfied by coated substrates which have orientated layers of proteins. Such substrates can be produced by immobilizing mutant proteins on a substrate--these mutant proteins have a marker in a desired and select spatial relationship with a preferential binding site, which binds the protein to functional groups of a substrate.